Structure of the bone morphogenetic protein receptor ALK2 and implications for fibrodysplasia ossificans progressiva

Abstract

Bone morphogenetic protein (BMP) receptor kinases are tightly regulated to control development and tissue homeostasis. Mutant receptor kinase domains escape regulation leading to severely degenerative diseases and represent an important therapeutic target. Fibrodysplasia ossificans progressiva (FOP) is a rare but devastating disorder of extraskeletal bone formation. FOP-associated mutations in the BMP receptor ALK2 reduce binding of the inhibitor FKBP12 and promote leaky signaling in the absence of ligand. To establish structural mechanisms of receptor regulation and to address the effects of FOP mutation, we determined the crystal structure of the cytoplasmic domain of ALK2 in complex with the inhibitors FKBP12 and dorsomorphin. FOP mutations break critical interactions that stabilize the inactive state of the kinase, thereby facilitating structural rearrangements that diminish FKBP12 binding and promote the correct positioning of the glycine-serine-rich loop and αC helix for kinase activation. The balance of these effects accounts for the comparable activity of R206H and L196P. Kinase activation in the clinically benign mutant L196P is far weaker than R206H but yields equivalent signals due to the stronger interaction of FKBP12 with R206H. The presented ALK2 structure offers a valuable template for the further design of specific inhibitors of BMP signaling.

FIGURE 1.

FOP mutations induce gain of function. A, schematic representation of the ALK2 protein showing the domain organization. FOP mutations map to the cytoplasmic GS and kinase domains. B, BRE-luciferase reporter assay for constitutive ALK2 activity. C2C12 cells were either left untreated (white bars), treated with 1 μm FK506 (light gray bars), or 10 ng/ml BMP4 (dark gray bars). y axis displays the ratio of Firefly/Renilla activity for transfected ALK2 constructs relative to vector control. Error bars represent standard error of at least three independent experiments performed in triplicate. In comparison with the untreated cells, FK506 significantly enhanced signaling by WT, R206H, G328E, G328R, and G356D (asterisk indicates significance at p < 0.05), whereas L196P, P197_F198delinsL, R202I, and Q207D were little affected (p > 0.6; ns indicates not significant). The remaining samples had intermediate p values (R258S and R375P were p < 0.1). Statistical analyses indicated no significant difference between FK506-treated FOP mutant samples and untreated caALK2 (p values >0.1). Additional data determined in parallel for BMP6 are shown in supplemental Fig. S1. C, representative expression levels for transfected ALK2 constructs. Control represents an endogenous protein bound by the anti-FLAG antibody. Numbers below indicate the band intensities for anti-FLAG normalized to control and shown relative to wild type.

FIGURE 2.

FOP mutations reduce FKBP12 binding. A, immunoprecipitation (IP) to determine FKBP12 binding to ALK2 in C2C12 cells transfected with FLAG-tagged ALK2 and HA-tagged FKBP12. Cells were either left untreated or treated with 1 μm FK506 as indicated. Proteins were detected with the indicated antibodies. The amount of immunoprecipitated ALK2 in each lane was quantified, normalized to input, and displayed relative to wild type. B, immunoprecipitation to determine FKBP12 binding to ALK2 in HEK293 cells treated as in A. WB, Western blot.

FIGURE 3.

Structure of the ALK2-FKBP12 complex. A, secondary structure elements are labeled and shown as ribbons. Disordered parts of the L45 loop and A loop are indicated by a thin dashed line. Dorsomorphin is shown in gray stick representation, and Mg²⁺ ion (cyan) and sulfate molecules (purple) are displayed as spheres. Inset box shows the specific interactions of dorsomorphin (yellow sticks) with the ATP pocket in ALK2. Hydrogen bonds (blue dashed lines) are formed with the hinge amide of His-286 and via a water molecule to Glu-248 (αC). The planarity of the phenyl ring is restricted by the close packing of Val-214 and Gly-289 (dashed gray lines). B, FKBP12 binding is dominated by insertion of the αGS2 helix into the central FK506-binding pocket of FKBP12. Here, the ALK5 residues Leu-195–Leu-196 are replaced by ALK2 Phe-198–Leu-199 resulting in a subtle shift in the complex interface. The hydrophobic contact surface in FKBP12 is colored yellow. C, |F_o| − |F_c| omit map contoured at 3σ (green mesh) for the bound dorsomorphin ligand (yellow sticks).

FIGURE 4.

Structural changes required for ALK2 activation. A, ALK2 (cyan, kinase; yellow, GS domain) adopts an inactive structure as shown by superposition with the type II BMP receptors ActRIIB (purple; Protein Data Bank 2QLU) or BMPRII (orange; Protein Data Bank 3G2F). Inset boxes and arrows highlight the movements required for activation of ALK2 (back view, top; front view, bottom). B, specific side chain interactions from the top inset box in A are shown. Here, the ALK2 GS loop adopts a buried conformation that is stabilized by interaction with Arg-258. C, specific side chain interactions from the lower inset box in A are shown. Here, ATP binding is sterically inhibited in ALK2 by the conformations of Tyr-219 (P loop) and Arg-375 (A loop).

FIGURE 5.

FOP mutations destabilize the inactive state. A, FOP mutation sites are clustered around the regulatory GS domain and ATP pocket. B, αGS2 helix in ALK2 is a hot spot for FOP mutation. Inset shows the likely charge repulsion between Q207E and FKBP12 Glu-55. C, structural models show that FOP mutations break inhibitory interactions in the ALK2-FKBP12 structure. Changes in the mutant structure are colored purple and overlaid onto the wild-type residue colored yellow. Hydrogen bonds in the wild type (yellow) and clashes in the mutant structure (purple) are shown by spheres and dashed lines, respectively. Models were generated using the Eris server (49).

FIGURE 6.

Schematic for ALK2 activation in FOP. The GS (yellow) and kinase (gray) domains are in equilibrium between inactive (left) and active (right) conformations. Top, in wild-type ALK2, FOP-associated residues (green sticks; Arg-206, Arg-258, and Arg-375) form critical interactions that maintain inhibitory conformations of the GS loop (purple), αC helix (blue), and A loop (red). The equilibrium is therefore shifted to the left, promoting binding of FKBP12. Bottom, in the R206H mutant, inhibitory GS-kinase domain interactions are broken, and the equilibrium is shifted to the right. FKBP12 is dissociated allowing interactions with the type II receptor (data not shown) (50). Opening of the GS loop and ATP pockets facilitates Smad assembly and phosphorylation.